CN106554405A

CN106554405A - A kind of polypeptide and application thereof, preparation method

Info

Publication number: CN106554405A
Application number: CN201510641027.3A
Authority: CN
Inventors: 朱日成; 陈军; 宓鹏程; 陶安进; 袁建成
Original assignee: Hybio Pharmaceutical Co Ltd
Current assignee: Hybio Pharmaceutical Co Ltd
Priority date: 2015-09-30
Filing date: 2015-09-30
Publication date: 2017-04-05
Anticipated expiration: 2035-09-30
Also published as: CN106554405B

Abstract

The present invention relates to Peptides Synthesis, more particularly to a kind of polypeptide and application thereof, preparation method.The polypeptide have (I), (II), (III) shown in aminoacid sequence in any one：(I), the adorned aminoacid sequence of aminoacid of the 1st, the 9th, the 20th, the 30th, the 34th and/or the 36th of Pramlintide；(II), the aminoacid sequence that one or several aminoacid are obtained is substituted, lacks or adds with the aminoacid sequence as shown in (I)；, and the sequence of (I) or (II) with least 70% homology (III).Pharmacokinetic property of the Pramlintide derivant of the present invention with prolongation and preferably pharmacodynamic properties.Therefore, amylin derivatives of the present invention need not be continually injected as existing pramlintide acetate.The inventive method is novel, synthesis condition is gentle, process is simple and process stabilizing.

Description

A kind of polypeptide and application thereof, preparation method

Technical field

The present invention relates to Peptides Synthesis, more particularly to a kind of polypeptide and application thereof, preparation method.

Background technology

Amylin is a kind of peptide hormone secreted by β cells, and which participates in the control of post-prandial glycemia, can subtract Slow food is in intestinal absorption speed.Pramlintide (Pramlintide) is then a kind of amylin class of synthetic Like thing, the serine of the alanine of amylin 25,28 and 29 is replaced by it with proline, is filled Divide the physiological action for remaining amylin, change the physical property that people's amylin is easy to assemble, it is to avoid The formation of amyloid deposition.Pramlintide (structure is as shown in formula I) is ground by Amylin companies of the U.S. earliest System, in March, 2005 obtain FDA approval, it is adaptable to using insulin but poor blood glucose control patient auxiliary Treatment.

Formula I

With the improvement of living condition, increasing people suffers from diabetes and obesity.Diabetes are which The metabolic disorder of the part ability or total loss of middle utilization glucose.Clinical research finds, when Pulan woods When peptide is shared with insulin, weight in patients may be caused moderately to mitigate.Pramlintide can be used as 1 type and 2 types The adjuvant therapy medicaments of diabetes, are mainly used in alone insulin, and use in conjunction insulin and sulfonylurea Class medicine and/or metformin cannot still obtain the diabeticss of expected effect.Pramlintide can be with islets of langerhans Element is shared, but can not replace insulin.

Currently, it is less with regard to the preparation report of Pramlintide modified derivative.Patent CN103596973A Describe the polypeptide of the aminoacid sequence of Pramlintide analog, the pharmaceutical composition comprising these polypeptides with And these polypeptides as medicine, which adopts the conduct such as carbon 14, carbon 16,20 diacid of carbon 18 and carbon to take The purpose of drug effect prolongation is reached for base modification；Patent CN102197049A is then with 17- carboxyl heptadecanoyls Base amino, 19- carboxyl nonadecane acyl groups and 19- carboxyl heptadecane acyl aminos etc. are used as modification means.

Pramlintide acetate prepared by existing product technology, the absolute bioavailability of subcutaneous single dose injection For 30～40%, subcutaneous injection reaches peak concentration in 27 minutes.Half-life is shorter, need to inject 2～3 (masters daily It is administered before the meal), administration frequency is higher, and patient must bear the injection pain of daily 2～3 times, not be easily accepted by.

Therefore it provides a kind of modified Pramlintide, extends its half-life, administration frequency is reduced, increased The compliance of strong patient has important practical significance.

The content of the invention

In view of this, the present invention provides a kind of polypeptide and application thereof, preparation method.The one of present invention offer Modified Pramlintide is planted, has been extended the half-life, has been reduced administration frequency, enhanced patient's compliance.

In order to realize foregoing invention purpose, the present invention provides technical scheme below：

The invention provides a kind of polypeptide, appoint in its aminoacid sequence for having shown in (I), (II), (III) Meaning one：

(I), the 1st, the 9th, the 20th, the 30th, the 34th and/or the 36th of Pramlintide The adorned aminoacid sequence of aminoacid of position；

(II), it is substituted, lacks or adds one or several aminoacid with the aminoacid sequence as shown in (I) to obtain The aminoacid sequence for obtaining；

, and the sequence of (I) or (II) with least 70% homology (III).

In some specific embodiments of the present invention, the modification includes union joint and/or fatty acid.

The present invention some specific embodiments in, the union joint selected from γ Glu, Arg, γ Glu-Arg, γGlu-His、γGlu-His、Glu-Lys、Glu-Glu、Glu-Arg、γGlu-His-His、γGlu-Arg-His、 γGlu-His-Arg、γGlu-Glu-Arg、Glu-Glu-Arg、Glu-Lys-Arg、γGlu-Glu-His-His、 γ Glu-Glu-His-Arg, Glu-Glu-Arg-Glu or Glu-Glu-Glu-Glu.Wherein, γ is (such as formula III institute Show) mean that the carboxyl that reaction site is γ positions is connected with corresponding aminoacid；Most preferably, joint " Con " choosing From γ Glu；

Formula III.

In some specific embodiments of the present invention, (I) is with SEQIDNO:Ammonia shown in 1 Base acid sequence；

(I) structure is as shown in formula II：

Formula II

Wherein, R1～R7 modifications junctional complex, including union joint and fatty acid.

The present invention some specific embodiments in, it is described be substituted by replacement 1,2,3,4 Individual or 5 aminoacid；

The disappearance is 1,2,3,4 or 5 aminoacid of disappearance；

It is described to be added to addition 1,2,3,4,5,6,7,8,9 Or 10 aminoacid.

Present invention also offers application of the polypeptide in the medicine for the treatment of diabetes or obesity is prepared.

Present invention also offers a kind of DNA molecular of coding said polypeptide.

Present invention also offers a kind of recombinant vector, which contains the DNA molecular.

Present invention also offers a kind of host cell, comprising described recombinant vector.

Present invention also offers a kind of pharmaceutical preparation for treating diabetes and/or obesity, by the polypeptide and Pharmaceutically acceptable adjuvant composition.

The dosage form of the pharmaceutical preparation can be gel, injectable powder, aerosol, spray, liniment, film It is agent, patch, unguentum, ointment, rubber-emplastrum, water preparation, decoction, electuary, tablet, pill, slow Release agent, controlled release agent, powder, paste, liniment, lotion, liniment, ionophore, eye drop, Nasal drop, gargarism, Sublingual tablet, insufflation, suppository, aerosol, inhalant, fumicants, oral liquid, Oral tablet, injection, syrup, soft extract, medicated wine, powder, granule, pill, tablet, glue Wafer, enema or suppository.Any feasible dosage form is all within protection scope of the present invention, of the invention Here is not limited.

Present invention also offers a kind of preparation method of the polypeptide, comprises the following steps：

Step 1：Take Tyr and be connected preparation Tyr- resins with resin；

Step 2：According to Pramlintide peptide sequence, remaining 36 aminoacid is sequentially coupled；

Step 3：By Thr³⁶、Ser³⁴、Thr³⁰、Ser²⁰、Thr⁹Side chain and/or Lys¹Deprotection, Expose corresponding functional group's hydroxyl or aminoacid；

Step 4：By the union joint and functional group's hydroxyl or amino acid condensation, ester bond or amide are formed It is bonded；

Step 5：The fatty acid is condensed with the union joint, amide is formed bonded；

Step 6：After the 2nd and the cyclisation of the 7th cysteine, cracking, purification, turn salt, obtain final product.

In some specific embodiments of the present invention, in the step 1, Fmoc-Tyr (t are prepared Bu) substitution degree of-MBHAResin is 0.2～0.3mmol/g, preferred 0.25mmol/g.

In some specific embodiments of the present invention, in the step 2,36 amino are sequentially coupled Combination of the coupling agent that acid is adopted for HOBT/DIPCDI.

In some specific embodiments of the present invention, in the step 2,36 ammonia being sequentially coupled In base acid, raw material：Thr³⁶From Fmoc-Thr (TBDPS)-OH, Ser³⁴From Fmoc-Ser (T BDPS)-OH, Thr³⁰From Fmoc-Thr (TBDPS)-OH, Ser²⁰From Fmoc-Ser (T BDPS)-OH, Thr⁹From Fmoc-Thr (TBDPS)-OH, Lys¹From Boc-Lys (Fmo C)-OH or Fmoc-Lys (Boc)-OH；The alternative TBDPS of wherein TBDMS；

In some specific embodiments of the present invention, in the step 2, peptide sequence is coupled Thr³⁶、Se r³⁴、Thr³⁰、Ser²⁰、Thr⁹Or Lys¹When, can optional (optional one, or together, above-mentioned aminoacid When it is several), it is optimum, only to select any of which to be preferred；

In some specific embodiments of the present invention, in the step 2, peptide sequence is coupled Thr³⁶、Th r³⁰Or Thr⁹When, Fmoc-Thr (OtBu) is selected if Fmoc-Thr (TBDPS)-OH is not selected -OH；It is coupled Ser³⁴And Ser²⁰Shi Ruo does not select Fmoc-Ser (TBDPS)-OH, then from Fm oc-Ser(tBu)-OH。

In some specific embodiments of the present invention, in the step 2, except Thr³⁶、Ser³⁴、T hr³⁰、Ser²⁰、Thr⁹And Lys¹Outward, other the raw material aminoacid being sequentially coupled are：Fmoc-Asn³⁵ (Trt)-OH、Fmoc-Gly-³³OH、Fmoc-Val³²-OH、Fmoc-Asn³¹(Trt)-OH、F moc-Pro²⁹-OH、Fmoc-Pro²⁸-OH、Fmoc-Leu²⁷-OH、Fmoc-Ile²⁶-OH、Fmoc-Pro²⁵-OH、Fmoc-Gly²⁴-OH、Fmoc-Phe²³-OH、Fmoc-Asn²²(Trt)-OH、Fmoc-As n²¹(Trt)-OH、Fmoc-Ser¹⁹(tBu)-OH、Fmoc-His¹⁸(Trt)-OH、Fmoc-Val¹⁷- OH、Fmoc-Leu¹⁶-OH、Fmoc-Phe¹⁵-OH、Fmoc-Asn¹⁴(Trt)-OH、Fmoc-Ala¹ ³-OH、Fmoc-Leu¹²-OH、Fmoc-Arg¹¹(Pbf)-OH、Fmoc-Gln¹⁰(Trt)-OH、F moc-Ala⁸-OH、Fmoc-Cys⁷(Trt)-OH、Fmoc-Thr⁶(tBu)-OH、Fmoc-Ala⁵- OH、Fmoc-Thr⁴(tBu)-OH、Fmoc-Asn³(Trt)-OH and Fmoc-Cys²(Trt)-O H。

In some specific embodiments of the present invention, the step 3 specially remove Thr36, Ser34, The protection of the TBDPS (or TBDMS) or removing Lys1 of Thr30, Ser20, Thr9 side chain protected Base Fmoc, exposes corresponding functional group (hydroxyl or amino).

In some specific embodiments of the present invention, in the step 3, remove what TBDPS was adopted Reagent is TBAF (2～5 times of amounts)/THF, preferably by TBAF (3 times of amounts)/THF.

In some specific embodiments of the present invention, in the step 3, the examination that Fmoc is adopted is removed Agent is 20% hexahydropyridine/DMF solution.

The present invention some specific embodiments in, in the step 4, joint " Con " selected from γ Glu, Arg、γGlu-Arg、γGlu-His、γGlu-His、Glu-Lys、Glu-Glu、Glu-Arg、γGlu-His -His、γGlu-Arg-His、γGlu-His-Arg、γGlu-Glu-Arg、Glu-Glu-Arg、Glu-Lys-Ar G, γ Glu-Glu-His-His, γ Glu-Glu-His-Arg, Glu-Glu-Arg-Glu and Glu-Glu-Glu-Gl U, wherein γ (as shown in formula III) mean that the carboxyl that reaction site is γ positions is connected with corresponding aminoacid；It is optimum Choosing, joint " Con " is selected from γ Glu；

Formula III

In some specific embodiments of the present invention, in the step 4, using (1.2 times of HOBT Amount)/DIPCDI (1.2 times amount)/DMAP (0.1 times of amount) is used as coupling agent；

In some specific embodiments of the present invention, in the step 5, fatty acid " FA " is selected from Hard Fat Acid, lauric acid or Palmic acid, most preferably Palmic acid；

In some specific embodiments of the present invention, in the step 5, using (1.2 times of HOBT Amount)/DMAP (0.1 times amount)/DIPCDI (1.2 times of amounts) is used as coupling agent；

In some specific embodiments of the present invention, the DMF specially using elemental iodine is molten for step 6 2nd and the 7th cysteine is cyclized by liquid.

In some specific embodiments of the present invention, in the step 6, specifically, cyclisation conditions are Elemental iodine (5～10 times amount), 1～3 hour, most preferably elemental iodine (8 times of amounts), 2 hours.

In some specific embodiments of the present invention, cracking, purification in step 6, turn salt and be specially： Peptide chain is cut using lysate, and with ether precipitation, washing, after being dried, obtain Pramlintide derivant Thick peptide；Using reversed phase high performance liquid phase method purifies and separates, turn salt, it is freeze-dried to obtain Pramlintide derivant Fine peptide.

In some specific embodiments of the present invention, in the step 6, lysate used is TFA/E DT/TIS/H₂O=90：5：3：2 (V/V), consumption are 1g resins correspondence 10ml lysates, are reacted Time is 2 hours.

In some specific embodiments of the present invention, in the step 6, isolate and purify and adopt NOVA SEPRP-HPLC systems, wavelength 220nm, chromatographic column be the anti-phase C18 posts of 150 × 250mm, A It is mutually the aqueous solution of 0.1% trifluoroacetic acid, B phases are pure acetonitrile, and gradient is 5%～95% (A Phase Proportion), 60 minutes.It is using 0.1mol/L ammonium acetate aqueous solutions/acetonitrile system to turn salt mobile phase.

Particularly, the stearic acid, lauric acid or Palmic acid, respectively with formula IV, formula V, VI institute of formula Show structure：

Stearic acid

Formula IV

Lauric acid

Formula V

Palmic acid

Formula VI

Finally, the Pramlintide derivant fine peptide for obtaining through lyophilizing, can be but be not limited only to shown below Meaning structure：

Stearic acid-(γ Glu)-[Thr³⁶]-Pramlintide；As shown in formula VII：

Formula VII

Stearic acid-(γ Glu)-[Ser³⁴]-Pramlintide；As shown in formula VIII：

Formula VIII

Stearic acid-(γ Glu)-[Thr³⁰]-Pramlintide；As shown in formula Ⅸ：

Formula Ⅸ

Stearic acid-(γ Glu)-[Ser²⁰]-Pramlintide；As shown in formula Ⅹ：

Formula Ⅹ

Stearic acid-(γ Glu)-[Thr⁹]-Pramlintide；As shown in formula Ⅺ：

Formula Ⅺ

Stearic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide；As shown in formula Ⅻ：

Formula Ⅻ

Stearic acid-(γ Glu)-[Lys¹Main chain]-Pramlintide；As shown in Formula X III：

Formula X III

Lauric acid-(γ Glu)-[Thr³⁶]-Pramlintide；As shown in Formula X IV：

Formula X IV

Lauric acid-(γ Glu)-[Ser³⁴]-Pramlintide；As shown in Formula X V：

Formula X V

Lauric acid-(γ Glu)-[Thr³⁰]-Pramlintide；As shown in Formula X VI：

Formula X VI

Lauric acid-(γ Glu)-[Ser²⁰]-Pramlintide；As shown in Formula X VII：

Formula X VII

Lauric acid-(γ Glu)-[Thr⁹]-Pramlintide；As shown in Formula X VIII：

Formula X VIII

Lauric acid-(γ Glu)-[Lys¹Side chain]-Pramlintide；As shown in Formula X IX：

Formula X IX

Lauric acid-(γ Glu)-[Lys¹Main chain]-Pramlintide；As shown in Formula X X：

Formula X X

Palmic acid-(γ Glu)-[Thr³⁶]-Pramlintide；As shown in Formula X XI：

Formula X XI

Palmic acid-(γ Glu)-[Ser³⁴]-Pramlintide；As shown in Formula X XII：

Formula X XII

Palmic acid-(γ Glu)-[Thr³⁰]-Pramlintide；As shown in Formula X XIII：

Formula X XIII

Palmic acid-(γ Glu)-[Ser²⁰]-Pramlintide；As shown in Formula X XIV：

Formula X XIV

Palmic acid-(γ Glu)-[Thr⁹]-Pramlintide；As shown in Formula X XV：

Formula X XV

Palmic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide；As shown in Formula X XVI：

Formula X XVI

Palmic acid-(γ Glu)-[Lys¹Main chain]-Pramlintide；As shown in Formula X XVII：

Formula X XVII

Stearic acid-(γ Glu-Arg)-[Thr³⁶]-Pramlintide；As shown in Formula X XVIII：

Formula X XVIII

Stearic acid-(γ Glu-Arg)-[Ser³⁴]-Pramlintide；As shown in Formula X XIX：

Formula X XIX

Stearic acid-(γ Glu-Arg)-[Thr³⁰]-Pramlintide；As shown in Formula X XX：

Formula X XX

Stearic acid-(γ Glu-Arg)-[Ser²⁰]-Pramlintide；As shown in Formula X XXI：

Formula X XXI

Stearic acid-(γ Glu-Arg)-[Thr⁹]-Pramlintide；As shown in Formula X XXII：

Formula X XXII

Stearic acid-(γ Glu-Arg)-[Lys¹Side chain]-Pramlintide；As shown in Formula X XXIII：

Formula X XXIII

Stearic acid-(γ Glu-Arg)-[Lys¹Main chain]-Pramlintide；As shown in Formula X XXIV：

Formula X XXIV

Lauric acid-(γ Glu-Arg)-[Thr³⁶]-Pramlintide；As shown in Formula X XXV：

Formula X XXV

Lauric acid-(γ Glu-Arg)-[Ser³⁴]-Pramlintide；As shown in Formula X XXVI：

Formula X XXVI

Lauric acid-(γ Glu-Arg)-[Thr³⁰]-Pramlintide；As shown in Formula X XXVII：

Formula X XXVII

Lauric acid-(γ Glu-Arg)-[Ser²⁰]-Pramlintide；As shown in Formula X XXVIII：

Formula X XXVIII

Lauric acid-(γ Glu-Arg)-[Thr⁹]-Pramlintide；As shown in Formula X XXIX：

Formula X XXIX

Lauric acid-(γ Glu-Arg)-[Lys¹Side chain]-Pramlintide；As shown in Formula X L：

Formula X L

Lauric acid-(γ Glu-Arg)-[Lys¹Main chain]-Pramlintide；As shown in Formula X LI：

Formula X LI

Palmic acid-(γ Glu-Arg)-[Thr³⁶]-Pramlintide；As shown in Formula X LII：

Formula X LII

Palmic acid-(γ Glu-Arg)-[Ser³⁴]-Pramlintide；As shown in Formula X LIII：

Formula X LIII

Palmic acid-(γ Glu-Arg)-[Thr³⁰]-Pramlintide；As shown in Formula X LIV：

Formula X LIV

Palmic acid-(γ Glu-Arg)-[Ser²⁰]-Pramlintide；As shown in Formula X LV：

Formula X LV

Palmic acid-(γ Glu-Arg)-[Thr⁹]-Pramlintide；As shown in Formula X LVI：

Formula X LVI

Palmic acid-(γ Glu-Arg)-[Lys¹Side chain]-Pramlintide；As shown in Formula X LVII：

Formula X LVII

Palmic acid-(γ Glu-Arg)-[Lys¹Main chain]-Pramlintide；As shown in Formula X LVIII：

Formula X LVIII

Palmic acid-(Arg)-[Lys¹Side chain]-Pramlintide；As shown in Formula X LIX：

Formula X LIX

Palmic acid-(γ Glu-His)-[Lys¹Side chain]-Pramlintide；As shown in formula L：

Formula L

Palmic acid-(Glu-Lys)-[Lys¹Side chain]-Pramlintide；As shown in formula LI：

Formula LI

Palmic acid-(Glu-Glu)-[Lys¹Side chain]-Pramlintide；As shown in formula LII：

Formula LII

Palmic acid-(Glu-Arg)-[Lys¹Side chain]-Pramlintide；As shown in formula LII：

Formula LII

Palmic acid-(γ Glu-His-His)-[Lys¹Side chain]-Pramlintide；As shown in formula LIII：

Formula LIII

Palmic acid-(γ Glu-Arg-His)-[Lys¹Side chain]-Pramlintide；As shown in formula LIV：

Formula LIV

Palmic acid-(γ Glu-His-Arg)-[Lys¹Side chain]-Pramlintide；As shown in formula LV：

Formula LV

Palmic acid-(γ Glu-Glu-Arg)-[Lys¹Side chain]-Pramlintide；As shown in formula LVI：

Formula LVI

Palmic acid-(Glu-Glu-Arg)-[Lys¹Side chain]-Pramlintide；As shown in formula LVII：

Formula LVII

Palmic acid-(Glu-Lys-Arg)-[Lys¹Side chain]-Pramlintide；As shown in formula LVIII：

Formula LVIII

Palmic acid-(γ Glu-Glu-His-His)-[Lys¹Side chain]-Pramlintide；As shown in formula LIX：

Formula LIX

Palmic acid-(γ Glu-Glu-His-Arg)-[Lys¹Side chain]-Pramlintide；As shown in formula LX：

Formula LX

Palmic acid-(Glu-Glu-Arg-Glu)-[Lys¹Side chain]-Pramlintide；As shown in formula LXI：

Formula LXI

Palmic acid-(Glu-Glu-Glu-Glu)-[Lys¹Side chain]-Pramlintide；As shown in formula LXII：

Formula LXII

Stearic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide-[Thr³⁰]-(γ Glu)-stearic acid；Such as formula Shown in LXIII：

Formula LXIII

Lauric acid-(γ Glu)-[Lys¹Side chain]-Pramlintide-[Thr³⁰]-(γ Glu)-lauric acid；Such as formula LXIV It is shown：

Formula LXIV

Palmic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide-[Thr³⁰]-(γ Glu)-Palmic acid；Such as formula Shown in LXV：

Formula LXV

Wherein, be union joint in bracket " () ", in which the 1st, left side amino acid active functional group with it is corresponding Specific amino acids active function groups connection in Pramlintide, γ represent that connection site is γ carboxyls.The right in which 1st aminoacid and the condensation of stearic acid, lauric acid or Palmic acid carboxyl；It is Pramlintide in bracket " [] " On designated amino acid, its active function groups and joint are condensed.

Preferably, preparing gained Pramlintide derivant is：

Stearic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide；

Stearic acid-(γ Glu)-[Lys¹Main chain]-Pramlintide；

Lauric acid-(γ Glu)-[Lys¹Side chain]-Pramlintide；

Lauric acid-(γ Glu)-[Lys¹Main chain]-Pramlintide；

Palmic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide；

Palmic acid-(γ Glu)-[Lys¹Main chain]-Pramlintide；

Most preferably, preparing gained Pramlintide derivant is：

Stearic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide；

Lauric acid-(γ Glu)-[Lys¹Side chain]-Pramlintide；

Palmic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide.

The invention provides a kind of polypeptide, appoint in its aminoacid sequence for having shown in (I), (II), (III) Meaning one：(I), the 1st, the 9th, the 20th, the 30th, the 34th of Pramlintide and/ Or the adorned aminoacid sequence of aminoacid of the 36th；(II), with the aminoacid sequence as shown in (I) It is substituted, lacks or adds the aminoacid sequence that one or several aminoacid are obtained；, and (I) or (II) (III) Sequence with least 70% homology.Pramlintide derivant of the present invention has the pharmacokineticss for extending Property and preferably pharmacodynamic properties.Therefore, amylin derivatives of the present invention need not with it is existing Pramlintide acetate the same continually inject.The inventive method is novel, synthesis condition is gentle, technique is simple List and process stabilizing.

Description of the drawings

In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to reality Apply accompanying drawing to be used needed for example or description of the prior art to be briefly described.

Fig. 1 shows stearic acid-(γ Glu)-[Lys1 side chains]-Pramlintide fine peptide figure prepared by embodiment 2；

Fig. 2 shows lauric acid-(γ Glu)-[Lys1 side chains]-Pramlintide fine peptide figure prepared by embodiment 3；

Fig. 3 shows Palmic acid-(γ Glu)-[Lys1 side chains]-Pramlintide fine peptide figure prepared by embodiment 4；

Fig. 4 shows stearic acid-(γ Glu)-[Lys prepared by embodiment 5¹Main chain]-Pramlintide fine peptide figure；

Fig. 5 shows lauric acid-(γ Glu)-[Lys prepared by embodiment 6¹Main chain]-Pramlintide fine peptide figure；

Fig. 6 shows Palmic acid-(γ Glu)-[Lys prepared by embodiment 7¹Main chain]-Pramlintide fine peptide figure；

Fig. 7 shows stearic acid-(γ Glu)-[Thr prepared by embodiment 8³⁰]-Pramlintide fine peptide figure；

Fig. 8 shows lauric acid-(γ Glu)-[Thr prepared by embodiment 9³⁰]-Pramlintide fine peptide figure；

Fig. 9 shows Palmic acid-(γ Glu)-[Thr prepared by embodiment 10³⁰]-Pramlintide fine peptide figure；

Figure 10 shows Palmic acid-(γ Glu-Arg)-[Thr prepared by embodiment 11³⁰]-Pramlintide fine peptide figure；

Figure 11 shows Palmic acid-(γ Glu)-[Lys prepared by embodiment 12¹Side chain]-Pramlintide-[Thr³⁰]- (γ Glu)-Palmic acid fine peptide figure；

Figure 12 shows that the Pramlintide and Symlin2 of subcutaneous injection present invention offer in embodiment 14 reaches 25 minutes Gastric content residual rate；

Figure 13 shows what the Symlin1 that Pramlintide in embodiment 15, the present invention provide and the present invention were provided Symlin2 blood sugar lowering Time-activity-curve figures.

Specific embodiment

The invention discloses a kind of polypeptide and application thereof, preparation method, those skilled in the art can use for reference Present disclosure, is suitably modified technological parameter realization.Specifically, all similar replacements and Change apparent to those skilled in the art, they are considered as being included in the present invention.This The method of invention and application are described by preferred embodiment, and related personnel can substantially not take off Method described herein and application are modified or are suitably changed in present invention, spirit and scope With combine, realize and apply the technology of the present invention.

In polypeptide that the present invention is provided and application thereof, preparation method, raw materials used and reagent can be purchased by market .

Abbreviation and English implication are shown in Table 1.

Table 1 is abridged and English implication

With reference to embodiment, the present invention is expanded on further：

1 substitution degree of embodiment is about the preparation of Fmoc-Tyr (the tBu)-MBHAResin of 0.25mmol/g

Take the RinkAmideMBHAResin resins that 2.5kg substitution degrees are 0.4mmol/g (common 1.0mol), in adding solid state reaction post, washed with DMF 2 times, with DMF swellable resins 30 minutes Afterwards, Fmoc is removed at twice using 20%DBLK solution, 10 minutes every time, then by resin DMF Washing 6 times.Take Fmoc-Tyr (tBu)-OH (0.8eq, 0.8mol, g), HOBt (0.96eq, 0.96mol, 130g) volume ratio is dissolved in DIPCDI (0.96eq, 0.96mol, 121g) for 1:1 DCM and DMF Mixed solution, in adding solid state reaction post, room temperature reaction 2h.Reaction is washed with DMF 2 times after terminating, DCM washes 2 times.It is subsequently adding pyridine (5eq, 5mol, 396g) and acetic anhydride (5eq, 5mol, 505g) Mixed liquor closing resin 6h (it is uniform that the appropriate DMF of addition makes it dispersed with stirring).4 are washed with DMF Secondary, after DCM washs 2 times, methanol shrinks to be drained, and obtains Fmoc-Tyr (tBu)-MBHAResin trees Fat, detection substitution degree are 0.251mmol/g, and weight is 2950g.

2 stearic acid of embodiment-(γ Glu)-[Lys¹Side chain]-Pramlintide preparation

The coupling of 2.1 aminoacid

398g is taken, Fmoc-Tyr (tBu)-MBHAResin resins prepared by 0.1mol embodiments 1 are added In solid state reaction post, washed with DMF 2 times, with DMF swellable resins 30 minutes after, using 20%DBLK Solution removes Fmoc at twice, 10 minutes every time, then resin is washed 6 times with DMF.Take Fmoc-Thr (tBu)-OH (5eq, 0.5mol, 199g), HOBt (6eq, 0.6mol, 81g) and DIPCDI (6eq, 0.6mol, 76g) is dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, add solid In phase reaction post, room temperature reaction 2h, with 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin water white transparency.Reaction Washed with DMF 3 times after end.

According to above-mentioned coupling mode, according to Pramlintide peptide sequence, Fmoc-Asn is coupled successively³⁵(Trt)-OH、 Fmoc-Ser³⁴(tBu)-OH、Fmoc-Gly-³³OH、Fmoc-Val³²-OH、Fmoc-Asn³¹(Trt) -OH、Fmoc-Thr³⁰(tBu)-OH、Fmoc-Pro²⁹-OH、Fmoc-Pro²⁸-OH、Fmoc-Leu²⁷-OH、 Fmoc-Ile²⁶-OH、Fmoc-Pro²⁵-OH、Fmoc-Gly²⁴-OH、Fmoc-Phe²³-OH、Fmoc-Asn²² (Trt)-OH、Fmoc-Asn²¹(Trt)-OH、Fmoc-Ser²⁰(tBu)-OH、Fmoc-Ser¹⁹(tBu) -OH、Fmoc-His¹⁸(Trt)-OH、Fmoc-Val¹⁷-OH、Fmoc-Leu¹⁶-OH、Fmoc-Phe¹⁵-OH、 Fmoc-Asn¹⁴(Trt)-OH、Fmoc-Ala¹³-OH、Fmoc-Leu¹²-OH、Fmoc-Arg¹¹(Pbf) -OH、Fmoc-Gln¹⁰(Trt)-OH、Fmoc-Thr⁹(tBu)-OH、Fmoc-Ala⁸-OH、Fmoc-Cys⁷ (Trt)-OH、Fmoc-Thr⁶(tBu)-OH、Fmoc-Ala⁵-OH、Fmoc-Thr⁴(tBu)-OH、 Fmoc-Asn³(Trt)-OH、Fmoc-Cys²(Trt)-OH and Boc-Lys¹(Fmoc)-OH。

Coupling is finished, and removes Fmoc at twice using 20%DBLK solution, 10 minutes every time, then will Resin DMF is washed 6 times, and Jing this processes, Boc-Lys¹(Fmoc)-OH side-chain amino groups are exposed Come.

The connection of the exposed Fmoc-Glu-OtBu of 2.2 side chain carboxyl groups and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (5eq, 0.5mol, 213g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, Volume ratio is dissolved in 76g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 2.1 solid state reaction post In, room temperature reaction 2h is colourless with 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent.Reaction is used after terminating DMF is washed 3 times.Fmoc is removed at twice with 20%DBLK solution, 10 minutes every time, then will tree Fat DMF washs 6 times.

2.3 stearic couplings

By stearic acid (5eq, 0.5mol, 142g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, 76g) are dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, in adding above-mentioned 2.2 solid state reaction post, room temperature reaction 3h uses 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent is colourless.Reaction washs 3 times after terminating with DMF.

The formation of 2.4 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 2.3 is added to solid In phase reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 903g's Stearic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide peptide resin.

2.5 stearic acid-(γ Glu)-[Lys¹Side chain] the thick peptide preparation of-Pramlintide

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. stearic acid-(γ Glu)-[Lys¹Side chain]- The thick peptide 531.2g of Pramlintide.It is 56.2% that weight yield is 106.2%, HPLC purity.

2.6 stearic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide preparation

Weigh 531.2g stearic acid-(γ Glu)-[Lys prepared by 2.5¹Side chain]-Pramlintide is thick After peptide is with 16L water dissolutioies, using NOVASEPRP-HPLC systems, wavelength 220nm, chromatographic column is The anti-phase C18 posts of 150 × 250mm, conventional 0.1%TFA/ acetonitrile mobile phases purification collect purpose peak fraction. Turn salt through ammonium acetate/acetonitrile system again, collect purpose peak fraction, rotary evaporation concentration, lyophilizing are obtained firmly Fat acid-(γ Glu)-[Lys¹Side chain]-Pramlintide fine peptide 212.5g, HPLC purity 99.93% is shown in figure 1。

3 lauric acid of embodiment-(γ Glu)-[Lys¹Side chain]-Pramlintide preparation

The coupling of 3.1 aminoacid

The connection of the exposed Fmoc-Glu-OtBu of 3.2 side chain carboxyl groups and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (5eq, 0.5mol, 213g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, Volume ratio is dissolved in 76g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 3.1 solid state reaction post In, room temperature reaction 2h is colourless with 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent.Reaction is used after terminating DMF is washed 3 times.Fmoc is removed at twice with 20%DBLK solution, 10 minutes every time, then will tree Fat DMF washs 6 times.

3.3 lauric couplings

By lauric acid (5eq, 0.5mol, 100g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, 76g) are dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, in adding above-mentioned 3.2 solid state reaction post, room temperature reaction 3h uses 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent is colourless.Reaction washs 3 times after terminating with DMF.

The formation of 3.4 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 2.3 is added to solid In phase reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 895g's Lauric acid-(γ Glu)-[Lys¹Side chain]-Pramlintide peptide resin.

3.5 lauric acids-(γ Glu)-[Lys¹Side chain] the thick peptide preparation of-Pramlintide

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. lauric acid-(γ Glu)-[Lys¹Side chain]- The thick peptide 516.9g of Pramlintide.It is 57.1% that weight yield is 103.4%, HPLC purity.

3.6 lauric acids-(γ Glu)-[Lys¹Side chain]-Pramlintide preparation

Weigh 516.9g lauric acids-(γ Glu)-[Lys prepared by 3.5¹Side chain]-Pramlintide is thick After peptide is with 16L water dissolutioies, using NOVASEPRP-HPLC systems, wavelength 220nm, chromatographic column is The anti-phase C18 posts of 150 × 250mm, conventional 0.1%TFA/ acetonitrile mobile phases purification collect purpose peak fraction. Turn salt through ammonium acetate/acetonitrile system again, collect purpose peak fraction, rotary evaporation concentration, lyophilizing obtain the moon Cinnamic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide fine peptide 209.2g, HPLC purity 99.92% is shown in figure 2。

4 Palmic acid of embodiment-(γ Glu)-[Lys¹Side chain]-Pramlintide preparation

The coupling of 4.1 aminoacid

The connection of the exposed Fmoc-Glu-OtBu of 4.2 side chain carboxyl groups and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (5eq, 0.5mol, 213g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, Volume ratio is dissolved in 76g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 4.1 solid state reaction post In, room temperature reaction 2h is colourless with 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent.Reaction is used after terminating DMF is washed 3 times.Fmoc is removed at twice with 20%DBLK solution, 10 minutes every time, then will tree Fat DMF washs 6 times.

The coupling of 4.3 Palmic acids

By Palmic acid (5eq, 0.5mol, 128g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, 76g) are dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, in adding above-mentioned 4.2 solid state reaction post, room temperature reaction 3h uses 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent is colourless.Reaction washs 3 times after terminating with DMF.

The formation of 4.4 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 4.3 is added to solid In phase reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 903g's Palmic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide peptide resin.

4.5 Palmic acids-(γ Glu)-[Lys¹Side chain] the thick peptide preparation of-Pramlintide

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. Palmic acid-(γ Glu)-[Lys¹Side chain]- The thick peptide 526.7g of Pramlintide.It is 56.7% that weight yield is 105.3%, HPLC purity.

4.6 Palmic acids-(γ Glu)-[Lys¹Side chain]-Pramlintide preparation

Weigh 526.7g Palmic acids-(γ Glu)-[Lys prepared by 2.5¹Side chain]-Pramlintide is thick After peptide is with 16L water dissolutioies, using NOVASEPRP-HPLC systems, wavelength 220nm, chromatographic column is The anti-phase C18 posts of 150 × 250mm, conventional 0.1%TFA/ acetonitrile mobile phases purification collect purpose peak fraction. Turn salt through ammonium acetate/acetonitrile system again, collect purpose peak fraction, rotary evaporation concentration, lyophilizing obtain palm fibre Palmitic acid acid-(γ Glu)-[Lys¹Side chain]-Pramlintide fine peptide 219.5g, HPLC purity 99.65% is shown in figure 3。

5 stearic acid of embodiment-(γ Glu)-[Lys¹Main chain]-Pramlintide

The coupling of 5.1 aminoacid

According to above-mentioned coupling mode, according to Pramlintide peptide sequence, Fmoc-Asn is coupled successively³⁵(Trt)-OH、 Fmoc-Ser³⁴(tBu)-OH、Fmoc-Gly-³³OH、Fmoc-Val³²-OH、Fmoc-Asn³¹(Trt) -OH、Fmoc-Thr³⁰(tBu)-OH、Fmoc-Pro²⁹-OH、Fmoc-Pro²⁸-OH、Fmoc-Leu²⁷-OH、 Fmoc-Ile²⁶-OH、Fmoc-Pro²⁵-OH、Fmoc-Gly²⁴-OH、Fmoc-Phe²³-OH、Fmoc-Asn²² (Trt)-OH、Fmoc-Asn²¹(Trt)-OH、Fmoc-Ser²⁰(tBu)-OH、Fmoc-Ser¹⁹(tBu) -OH、Fmoc-His¹⁸(Trt)-OH、Fmoc-Val¹⁷-OH、Fmoc-Leu¹⁶-OH、Fmoc-Phe¹⁵-OH、 Fmoc-Asn¹⁴(Trt)-OH、Fmoc-Ala¹³-OH、Fmoc-Leu¹²-OH、Fmoc-Arg¹¹(Pbf) -OH、Fmoc-Gln¹⁰(Trt)-OH、Fmoc-Thr⁹(tBu)-OH、Fmoc-Ala⁸-OH、Fmoc-Cys⁷ (Trt)-OH、Fmoc-Thr⁶(tBu)-OH、Fmoc-Ala⁵-OH、Fmoc-Thr⁴(tBu)-OH、 Fmoc-Asn³(Trt)-OH、Fmoc-Cys²(Trt)-OH and Fmoc-Lys¹(Boc)-OH。

Coupling is finished, and removes Fmoc at twice using 20%DBLK solution, 10 minutes every time, then will Resin DMF is washed 6 times, and Jing this processes, Fmoc-Lys¹(Boc)-OH backbone aminos are exposed Come.

The connection of the exposed Fmoc-Glu-OtBu of 5.2 side chain carboxyl groups and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (5eq, 0.5mol, 213g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, Volume ratio is dissolved in 76g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 5.1 solid state reaction post In, room temperature reaction 2h is colourless with 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent.Reaction is used after terminating DMF is washed 3 times.Fmoc is removed at twice with 20%DBLK solution, 10 minutes every time, then will tree Fat DMF washs 6 times.

5.3 stearic couplings

By stearic acid (5eq, 0.5mol, 142g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, 76g) are dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, in adding above-mentioned 5.2 solid state reaction post, room temperature reaction 3h uses 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent is colourless.Reaction washs 3 times after terminating with DMF.

The formation of 5.4 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 2.3 is added to solid In phase reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 907g's Stearic acid-(γ Glu)-[Lys¹Main chain]-Pramlintide peptide resin.

5.5 stearic acid-(γ Glu)-[Lys¹Main chain] the thick peptide preparation of-Pramlintide

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. stearic acid-(γ Glu)-[Lys¹Main chain]- The thick peptide 530.2g of Pramlintide.It is 56.8% that weight yield is 106.0%, HPLC purity.

5.6 stearic acid-(γ Glu)-[Lys¹Main chain]-Pramlintide preparation

Weigh 530.2g stearic acid-(γ Glu)-[Lys prepared by 5.5¹Main chain]-Pramlintide is thick After peptide is with 16L water dissolutioies, using NOVASEPRP-HPLC systems, wavelength 220nm, chromatographic column is The anti-phase C18 posts of 150 × 250mm, conventional 0.1%TFA/ acetonitrile mobile phases purification collect purpose peak fraction. Turn salt through ammonium acetate/acetonitrile system again, collect purpose peak fraction, rotary evaporation concentration, lyophilizing are obtained firmly Fat acid-(γ Glu)-[Lys¹Main chain]-Pramlintide fine peptide 213.2g, HPLC purity 99.2%.

6 lauric acid of embodiment-(γ Glu)-[Lys¹Main chain]-Pramlintide preparation

The coupling of 6.1 aminoacid

The connection of the exposed Fmoc-Glu-OtBu of 6.2 side chain carboxyl groups and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (5eq, 0.5mol, 213g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, Volume ratio is dissolved in 76g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 6.1 solid state reaction post In, room temperature reaction 2h is colourless with 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent.Reaction is used after terminating DMF is washed 3 times.Fmoc is removed at twice with 20%DBLK solution, 10 minutes every time, then will tree Fat DMF washs 6 times.

6.3 lauric couplings

By lauric acid (5eq, 0.5mol, 100g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, 76g) are dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, in adding above-mentioned 6.2 solid state reaction post, room temperature reaction 3h uses 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent is colourless.Reaction washs 3 times after terminating with DMF.

The formation of 6.4 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 6.3 is added to solid In phase reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 896g's Lauric acid-(γ Glu)-[Lys¹Main chain]-Pramlintide peptide resin.

6.5 lauric acids-(γ Glu)-[Lys¹Main chain] the thick peptide preparation of-Pramlintide

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. lauric acid-(γ Glu)-[Lys¹Main chain]- The thick peptide 512.2g of Pramlintide.It is 56.1% that weight yield is 102.4%, HPLC purity.

6.6 lauric acids-(γ Glu)-[Lys¹Main chain]-Pramlintide preparation

Weigh 512.2g lauric acids-(γ Glu)-[Lys prepared by 6.5¹Main chain]-Pramlintide is thick After peptide is with 16L water dissolutioies, using NOVASEPRP-HPLC systems, wavelength 220nm, chromatographic column is The anti-phase C18 posts of 150 × 250mm, conventional 0.1%TFA/ acetonitrile mobile phases purification collect purpose peak fraction. Turn salt through ammonium acetate/acetonitrile system again, collect purpose peak fraction, rotary evaporation concentration, lyophilizing obtain the moon Cinnamic acid-(γ Glu)-[Lys¹Main chain]-Pramlintide fine peptide 206g, HPLC purity 99.3%.

7 Palmic acid of embodiment-(γ Glu)-[Lys¹Main chain]-Pramlintide preparation

The coupling of 7.1 aminoacid

The connection of the exposed Fmoc-Glu-OtBu of 7.2 side chain carboxyl groups and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (5eq, 0.5mol, 213g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, Volume ratio is dissolved in 76g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 7.1 solid state reaction post In, room temperature reaction 2h is colourless with 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent.Reaction is used after terminating DMF is washed 3 times.Fmoc is removed at twice with 20%DBLK solution, 10 minutes every time, then will tree Fat DMF washs 6 times.

The coupling of 7.3 Palmic acids

By Palmic acid (5eq, 0.5mol, 128g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, 76g) are dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, in adding above-mentioned 7.2 solid state reaction post, room temperature reaction 3h uses 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent is colourless.Reaction washs 3 times after terminating with DMF.

The formation of 7.4 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 6.3 is added to solid In phase reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 912.1g Palmic acid-(γ Glu)-[Lys¹Main chain]-Pramlintide peptide resin.

7.5 Palmic acids-(γ Glu)-[Lys¹Main chain] the thick peptide preparation of-Pramlintide

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. Palmic acid-(γ Glu)-[Lys¹Main chain]- The thick peptide 527.2g of Pramlintide.It is 57.1% that weight yield is 105.4%, HPLC purity.

7.6 Palmic acids-(γ Glu)-[Lys¹Main chain]-Pramlintide preparation

Weigh 512.2g Palmic acids-(γ Glu)-[Lys prepared by 7.5¹Main chain]-Pramlintide is thick After peptide is with 16L water dissolutioies, using NOVASEPRP-HPLC systems, wavelength 220nm, chromatographic column is The anti-phase C18 posts of 150 × 250mm, conventional 0.1%TFA/ acetonitrile mobile phases purification collect purpose peak fraction. Turn salt through ammonium acetate/acetonitrile system again, collect purpose peak fraction, rotary evaporation concentration, lyophilizing obtain palm fibre Palmitic acid acid-(γ Glu)-[Lys¹Main chain]-Pramlintide fine peptide 217g, HPLC purity 99.4%.

8 stearic acid of embodiment-(γ Glu)-[Thr³⁰The preparation of]-Pramlintide

The coupling of 8.1 aminoacid

According to above-mentioned coupling mode, according to Pramlintide peptide sequence, Fmoc-Asn is coupled successively³⁵(Trt)-OH、 Fmoc-Ser³⁴(tBu)-OH、Fmoc-Gly-³³OH、Fmoc-Val³²-OH、Fmoc-Asn³¹(Trt) -OH、Fmoc-Thr³⁰(TBDPS)-OH、Fmoc-Pro²⁹-OH、Fmoc-Pro²⁸-OH、 Fmoc-Leu²⁷-OH、Fmoc-Ile²⁶-OH、Fmoc-Pro²⁵-OH、Fmoc-Gly²⁴-OH、 Fmoc-Phe²³-OH、Fmoc-Asn²²(Trt)-OH、Fmoc-Asn²¹(Trt)-OH、Fmoc-Ser²⁰ (tBu)-OH、Fmoc-Ser¹⁹(tBu)-OH、Fmoc-His¹⁸(Trt)-OH、Fmoc-Val¹⁷-OH、 Fmoc-Leu¹⁶-OH、Fmoc-Phe¹⁵-OH、Fmoc-Asn¹⁴(Trt)-OH、Fmoc-Ala¹³-OH、 Fmoc-Leu¹²-OH、Fmoc-Arg¹¹(Pbf)-OH、Fmoc-Gln¹⁰(Trt)-OH、Fmoc-Thr⁹ (tBu)-OH、Fmoc-Ala⁸-OH、Fmoc-Cys⁷(Trt)-OH、Fmoc-Thr⁶(tBu)-OH、 Fmoc-Ala⁵-OH、Fmoc-Thr⁴(tBu)-OH、Fmoc-Asn³(Trt)-OH、Fmoc-Cys² (Trt)-OH and Boc-Lys¹(Boc)-OH。

Thr in 8.2 peptide chains³⁰Side chain protective group TBDPS removing

TBAF (3eq, 0.3mol, 78g) is dissolved in the THF of 1.0L, and is added it to State in 8.1 solid state reaction posts, air-blowing stirring reaction 3 hours.Reaction washs 3 times after terminating with DMF.

The connection of the exposed Fmoc-Glu-OtBu of 8.3 side chain carboxyl groups and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (5eq, 0.5mol, 213g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, Volume ratio is dissolved in 76g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 8.2 solid state reaction post In, room temperature reaction 3h.Reaction washs 3 times after terminating with DMF.With 20%DBLK solution at twice Removing Fmoc, 10 minutes every time, then resin is washed into 6 times with DMF.

8.4 stearic couplings

By stearic acid (5eq, 0.5mol, 142g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, 76g) are dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, in adding above-mentioned 8.3 solid state reaction post, room temperature reaction 3h uses 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent is colourless.Reaction washs 3 times after terminating with DMF.

The formation of 8.5 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 8.4 is added to solid In phase reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 938.7g Stearic acid-(γ Glu)-[Thr³⁰]-Pramlintide peptide resin.

8.6 stearic acid-(γ Glu)-[Thr³⁰It is prepared by the thick peptide of]-Pramlintide

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. stearic acid-(γ Glu)-[Thr³⁰]-general The thick peptide 537.3g of blue woods peptide.It is 57.2% that weight yield is 107.4%, HPLC purity.

8.7 stearic acid-(γ Glu)-[Thr³⁰The preparation of]-Pramlintide

Weigh 537.3g stearic acid-(γ Glu)-[Thr prepared by 8.6³⁰The thick peptide of]-Pramlintide is used After 16L water dissolutioies, using NOVASEPRP-HPLC systems, wavelength 220nm, chromatographic column are 150 × 250 The anti-phase C18 posts of mm, conventional 0.1%TFA/ acetonitrile mobile phases purification collect purpose peak fraction.Pass through again Ammonium acetate/acetonitrile system turns salt, collects purpose peak fraction, rotary evaporation concentration, lyophilizing obtain stearic acid- (γGlu)-[Thr³⁰]-Pramlintide fine peptide 227g, HPLC purity 99.4%.

Embodiment September cinnamic acid-(γ Glu)-[Thr³⁰The preparation of]-Pramlintide

The coupling of 9.1 aminoacid

Thr in 9.2 peptide chains³⁰Side chain protective group TBDPS removing

TBAF (3eq, 0.3mol, 78g) is dissolved in the THF of 1.0L, and is added it to State in 9.1 solid state reaction posts, air-blowing stirring reaction 3 hours.Reaction washs 3 times after terminating with DMF.

The connection of the exposed Fmoc-Glu-OtBu of 9.3 side chain carboxyl groups and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (5eq, 0.5mol, 213g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, Volume ratio is dissolved in 76g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 9.2 solid state reaction post In, room temperature reaction 3h.Reaction washs 3 times after terminating with DMF.With 20%DBLK solution at twice Removing Fmoc, 10 minutes every time, then resin is washed into 6 times with DMF.

9.4 lauric couplings

By lauric acid (5eq, 0.5mol, 100g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, 76g) are dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, in adding above-mentioned 9.3 solid state reaction post, room temperature reaction 3h uses 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent is colourless.Reaction washs 3 times after terminating with DMF.

The formation of 9.5 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 9.4 is added to solid In phase reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 921.2g Lauric acid-(γ Glu)-[Thr³⁰]-Pramlintide peptide resin.

9.6 lauric acids-(γ Glu)-[Thr³⁰It is prepared by the thick peptide of]-Pramlintide

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. lauric acid-(γ Glu)-[Thr³⁰]-general The thick peptide 527.3g of blue woods peptide.It is 56.8% that weight yield is 105.4%, HPLC purity.

9.7 lauric acids-(γ Glu)-[Thr³⁰The preparation of]-Pramlintide

Weigh 527.3g lauric acids-(γ Glu)-[Thr prepared by 9.6³⁰The thick peptide of]-Pramlintide is used After 16L water dissolutioies, using NOVASEPRP-HPLC systems, wavelength 220nm, chromatographic column are 150 × 250 The anti-phase C18 posts of mm, conventional 0.1%TFA/ acetonitrile mobile phases purification collect purpose peak fraction.Pass through again Ammonium acetate/acetonitrile system turns salt, collects purpose peak fraction, rotary evaporation concentration, lyophilizing obtain lauric acid- (γGlu)-[Thr³⁰]-Pramlintide fine peptide 217g, HPLC purity 99.3%.

10 Palmic acid of embodiment-(γ Glu)-[Thr³⁰The preparation of]-Pramlintide

The coupling of 10.1 aminoacid

Thr in 10.2 peptide chains³⁰Side chain protective group TBDPS removing

TBAF (3eq, 0.3mol, 78g) is dissolved in the THF of 1.0L, and is added it to State in 10.1 solid state reaction posts, air-blowing stirring reaction 3 hours.Reaction washs 3 times after terminating with DMF.

The connection of the exposed Fmoc-Glu-OtBu of 10.3 side chain carboxyl groups and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (5eq, 0.5mol, 213g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, Volume ratio is dissolved in 76g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 10.2 solid state reaction In post, room temperature reaction 3h.Reaction washs 3 times after terminating with DMF.It is divided to two with 20%DBLK solution Secondary removing Fmoc, 10 minutes every time, then resin is washed into 6 times with DMF.

The coupling of 10.4 Palmic acids

By Palmic acid (5eq, 0.5mol, 124g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, 76g) are dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, in adding above-mentioned 10.3 solid state reaction post, room temperature reaction 3h is used 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent is colourless.Reaction washs 3 times after terminating with DMF.

The formation of 10.5 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 10.4 are added to In solid state reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 928.7g Palmic acid-(γ Glu)-[Thr³⁰]-Pramlintide peptide resin.

10.6 Palmic acids-(γ Glu)-[Thr³⁰It is prepared by the thick peptide of]-Pramlintide

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. Palmic acid-(γ Glu)-[Thr³⁰]-general The thick peptide 529.3g of blue woods peptide.It is 57.1% that weight yield is 105.8%, HPLC purity.

10.7 Palmic acids-(γ Glu)-[Thr³⁰The preparation of]-Pramlintide

Weigh 529.3g Palmic acids-(γ Glu)-[Thr prepared by 10.6³⁰The thick peptide of]-Pramlintide After with 16L water dissolutioies, using NOVASEPRP-HPLC systems, wavelength 220nm, chromatographic column is The anti-phase C18 posts of 150 × 250mm, conventional 0.1%TFA/ acetonitrile mobile phases purification collect purpose peak fraction. Turn salt through ammonium acetate/acetonitrile system again, collect purpose peak fraction, rotary evaporation concentration, lyophilizing obtain palm fibre Palmitic acid acid-(γ Glu)-[Thr³⁰]-Pramlintide fine peptide 223g, HPLC purity 99.3%.

11 Palmic acid of embodiment-(γ Glu-Arg)-[Thr³⁰The preparation of]-Pramlintide

The coupling of 11.1 aminoacid

Thr in 11.2 peptide chains³⁰Side chain protective group TBDPS removing

TBAF (3eq, 0.3mol, 78g) is dissolved in the THF of 1.0L, and is added it to State in 11.1 solid state reaction posts, air-blowing stirring reaction 3 hours.Reaction washs 3 times after terminating with DMF.

The connection of 11.3 joints " γ Glu-Arg " and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (5eq, 0.5mol, 213g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, Volume ratio is dissolved in 76g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 11.2 solid state reaction In post, room temperature reaction 3h.Reaction washs 3 times after terminating with DMF.It is divided to two with 20%DBLK solution Secondary removing Fmoc, 10 minutes every time, then resin is washed into 6 times with DMF.

Fmoc-Arg (Pbf)-OH will be coupled by above-mentioned coupling mode, and with 5% 1,2,3-indantrione monohydrate/ethanol solution Detection resin water white transparency.Reaction washs 3 times after terminating with DMF.With 20%DBLK solution at twice Removing Fmoc, 10 minutes every time, then resin is washed into 6 times with DMF.

The coupling of 11.4 Palmic acids

By Palmic acid (5eq, 0.5mol, 124g), HOBt (6eq, 0.6mol, 81g), DMAP (0.5eq, 0.05mol, 6.1g) and DIPCDI (6eq, 0.6mol, 76g) are dissolved in volume ratio for 1:1 DCM and DMF mixed solutions, in adding above-mentioned 11.3 solid state reaction post, room temperature reaction 3h is used 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent is colourless.Reaction washs 3 times after terminating with DMF.

The formation of 11.5 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 11.4 are added to In solid state reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 940.7g Palmic acid-(γ Glu-Arg)-[Thr³⁰]-Pramlintide peptide resin.

11.6 Palmic acids-(γ Glu-Arg)-[Thr³⁰It is prepared by the thick peptide of]-Pramlintide

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. Palmic acid-(γ Glu-Arg)-[Thr³⁰]- The thick peptide 532.3g of Pramlintide.It is 57.2% that weight yield is 106.4%, HPLC purity.

11.7 Palmic acids-(γ Glu-Arg)-[Thr³⁰The preparation of]-Pramlintide

Weigh 532.3g Palmic acids-(γ Glu-Arg)-[Thr prepared by 11.6³⁰]-Pramlintide is thick After peptide is with 16L water dissolutioies, using NOVASEPRP-HPLC systems, wavelength 220nm, chromatographic column is The anti-phase C18 posts of 150 × 250mm, conventional 0.1%TFA/ acetonitrile mobile phases purification collect purpose peak fraction. Turn salt through ammonium acetate/acetonitrile system again, collect purpose peak fraction, rotary evaporation concentration, lyophilizing obtain palm fibre Palmitic acid acid-(γ Glu-Arg)-[Thr³⁰]-Pramlintide fine peptide 219g, HPLC purity 99.4%.

12 Palmic acid of embodiment-(γ Glu)-[Lys¹Side chain]-Pramlintide-[Thr³⁰]-(γ Glu)-Palmic acid Prepare

The coupling of 12.1 aminoacid

According to above-mentioned coupling mode, according to Pramlintide peptide sequence, Fmoc-Asn is coupled successively³⁵(Trt)-OH、 Fmoc-Ser³⁴(tBu)-OH、Fmoc-Gly-³³OH、Fmoc-Val³²-OH、Fmoc-Asn³¹(Trt) -OH、Fmoc-Thr³⁰(TBDPS)-OH、Fmoc-Pro²⁹-OH、Fmoc-Pro²⁸-OH、 Fmoc-Leu²⁷-OH、Fmoc-Ile²⁶-OH、Fmoc-Pro²⁵-OH、Fmoc-Gly²⁴-OH、 Fmoc-Phe²³-OH、Fmoc-Asn²²(Trt)-OH、Fmoc-Asn²¹(Trt)-OH、Fmoc-Ser²⁰ (tBu)-OH、Fmoc-Ser¹⁹(tBu)-OH、Fmoc-His¹⁸(Trt)-OH、Fmoc-Val¹⁷-OH、 Fmoc-Leu¹⁶-OH、Fmoc-Phe¹⁵-OH、Fmoc-Asn¹⁴(Trt)-OH、Fmoc-Ala¹³-OH、 Fmoc-Leu¹²-OH、Fmoc-Arg¹¹(Pbf)-OH、Fmoc-Gln¹⁰(Trt)-OH、Fmoc-Thr⁹ (tBu)-OH、Fmoc-Ala⁸-OH、Fmoc-Cys⁷(Trt)-OH、Fmoc-Thr⁶(tBu)-OH、 Fmoc-Ala⁵-OH、Fmoc-Thr⁴(tBu)-OH、Fmoc-Asn³(Trt)-OH、Fmoc-Cys² (Trt)-OH and Boc-Lys¹(Fmoc)-OH。

Thr in 12.2 peptide chains³⁰Side chain protective group TBDPS removing

TBAF (3eq, 0.3mol, 78g) is dissolved in the THF of 1.0L, and is added into To in above-mentioned 12.1 solid state reaction post, air-blowing stirring reaction 3 hours.Reaction washs 3 with DMF after terminating It is secondary.

The connection of the exposed Fmoc-Glu-OtBu of 12.3 side chain carboxyl groups and Pramlintide (cyclisation)

By side chain carboxyl group exposed Fmoc-Glu-OtBu (10eq, 1.0mol, 426g), HOBt (12eq, 1.2mol, 162g), DMAP (1.0eq, 0.1mol, 12.2g) and DIPCDI (12eq, 1.2mol, Volume ratio is dissolved in 152g) for 1:1 DCM and DMF mixed solutions, add above-mentioned 12.2 solid state reaction In post, room temperature reaction 3h is colourless with 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent.After reaction terminates 3 times are washed with DMF.Fmoc is removed at twice with 20%DBLK solution, 10 minutes every time, then will Resin DMF washs 6 times.

The coupling of 12.4 Palmic acids

By Palmic acid (10eq, 1.0mol, 256g), HOBt (12eq, 1.2mol, 162g), DMAP (1.0eq, 0.1mol, 12.2g) and DIPCDI (12eq, 1.2mol, 152g) are dissolved in volume ratio and are 1:1 DCM and DMF mixed solutions, in adding above-mentioned 12.3 solid state reaction post, room temperature reaction 3h, It is colourless with 5% 1,2,3-indantrione monohydrate/ethanol solution detection resin transparent.Reaction washs 3 times after terminating with DMF.

The formation of 12.5 disulfide bond

Elemental iodine (8eq, 0.8mol, 203g) is dissolved in 4.0LDMF, above-mentioned 12.4 are added to In solid state reaction post, 25 DEG C of reaction 2h, after reaction terminates, wash 6 with 5% hexahydropyridine/DMF solution It is secondary, finally washed with DMF 4 times, after DCM washs 2 times, methanol shrinks to be drained, and obtains 956g's Palmic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide-[Thr³⁰]-(γ Glu)-Palmic acid peptide resin.

12.6 Palmic acids-(γ Glu)-[Lys¹Side chain]-Pramlintide-[Thr³⁰]-(γ Glu) the thick peptide system of-Palmic acid It is standby

Lytic reagent (TFA/EDT/TIS/H is added with the ratio of 10ml/g peptide resins₂O=90：5：3：2 (V/V) 2h is stirred at room temperature),.Reactant is filtered with sand core funnel, collects filtrate, and resin is again with less Amount TFA is washed 3 times, concentrating under reduced pressure after merging filtrate.The absolute ether precipitation of frost is added, with anhydrous Ether is washed 3 times, and vacuum drying obtains white powder solid, i.e. Palmic acid-(γ Glu)-[Lys¹Side chain]- Pramlintide-[Thr³⁰]-(γ Glu)-Palmic acid 553.7g.Weight yield is 108.4%, HPLC purity For 55.7%.

12.7 Palmic acids-(γ Glu)-[Lys¹Side chain]-Pramlintide-[Thr³⁰]-(γ Glu)-Palmic acid preparation

Weigh 553.7g Palmic acids-(γ Glu)-[Lys prepared by 12.6¹Side chain]-Pramlintide -[Thr³⁰]-(γ Glu) the thick peptide of-Palmic acid with 18L water dissolutioies after, using NOVASEPRP-HPLC systems System, wavelength 220nm, chromatographic column be the anti-phase C18 posts of 150 × 250mm, conventional 0.1%TFA/ acetonitriles stream Dynamic phase purification, collects purpose peak fraction.Turn salt through ammonium acetate/acetonitrile system again, collect purpose peak fraction, Rotary evaporation is concentrated, and lyophilizing obtains Palmic acid-(γ Glu)-[Lys1 side chains]-Pramlintide-[Thr30]-(γ Glu) - Palmic acid fine peptide 232.5g, HPLC purity 99.2%.

The detection of the external activity of the Pramlintide trim that 13 Pramlintide of embodiment and the present invention are provided

Pramlintide and its derivant are determined in culture using the L6 cells sugar absorbing model of Jing inductions The impact of the situation of change of glucose in clear.

L6 cell Jing inductions are formed into myotube, culture medium is removed, orifice plate adds 0.4ml DMEM in high glucose The hungry culture 2h of culture medium (containing 2%FBS), removes culture medium, three is prepared when equivalent means are common Group.Wherein two groups difference 400 μ L Pramlintide of accurate addition and Palmic acid-(γ Glu)-[Lys¹Side chain]- Pramlintide (to be easy to description, making as Symlin2) titer (is 1x10^-6M, solvent are 11mM Glucose KHHbuffer)；Another set is matched group, does not contain medicine, only contains 11mM Glucose KHHbuffer.Per group, respectively in 4h, 8h, 16h, 24h and 48h, takes 200 μ L per hole Culture fluid supernatant, takes supernatant and determines glucose content after centrifugation, the sugar for calculating cell absorbs incrementss and increasing Plus rate.As a result shown in table 2：

2 Pramlintide 1 of table and Symlin2 sugar absorb increment and sugar absorbs increment rate result table

As a result show, compared with matched group, from 4h to 48h, Pramlintide and Symlin2 can promote Absorption of the L6 cells to glucose, and absorbtivity increases over time and increases, particularly, In T=16h, cell sugar absorbs and gathers way most fast, and action time is short, also illustrate Pramlintide and Symlin2 has obvious external activity.Meanwhile, in T=48 hours, the sugar of Symlin2 absorbs and increases It is plus rate is 63.27 ± 1.205%, high compared with Pramlintide 45.25 ± 2.215%, with pole significant difference (P ＜ 0.01), show that the active duration of the performance of Symlin2 is relatively long.

Particularly, using this law, the part Pramlintide derivant prepared by the present invention body has been carried out into The detection of outer activity, and during by T=48 hours, the sugar of each derivant absorbs increment rate and is listed in table 3：

3 Pramlintide derivatives activity data table of table

Therefore, absorb increment rate data from sugar to draw, the Pramlintide derivant of Jing present invention modifications The duration extremely notable (P ＜ 0.01) of external activity is played better than Pramlintide.

The detection of the activity in vivo of the Pramlintide trim that 14 Pramlintide of embodiment and the present invention are provided

Research data shows that Pramlintide can play the work for reducing post-prandial glycemia by delaying gastric emptying With.The embodiment of the present invention can be hydrophilic swelling using macromolecular material methylcellulose and be inhaled inside intestinal Receive also without local irritant effect the characteristics of, prepare half mobility thick gel solution, while with it is phenol red as refer to Show agent to reflect indirectly gastric content residual quantity.

14.1 prepare gavage liquid

The methocel solution 100ml that preparation is added with phenol red 15mg/ml is standby；

14.2 experimental rats

Prepare the male and healthy rat of 225 ± 10g of body weight and feed one week, water 20h is can't help in fasting before experiment, And be classified as with the following group：

Blank control group：16

Positive controls：Pramlintide dosage be 0.15,0.75,1.5,15,150 μ g, 8 per group

Administration group：Symlin2 dosage be 0.15,0.75,1.5,15,150 μ g, 8 per group

14.3 administrations

According to the dosage (each 0.5ml) of 0.15,0.75,1.5,15,150 μ g, by positive controls and The rat of administration group injects Pramlintide and Symlin2 respectively, and blank control group administration equal-volume 0.9% is given birth to Reason saline.After 5 minutes, every rat adopts gavage liquid gavage 1.5ml.

14.4 content is collected

8 positive controls rats are taken, is anaesthetized after gavage immediately and is dissected collection gastric content；Remaining rat After gavage 20 minutes, then anaesthetize and dissect collection gastric content.

14.5 gastric content residual rates are determined

Will be through 0.5M sodium hydroxide (0.5ml) process, and the supernatant that obtains of Jing centrifuging and takings, in 560nm Lower measurement absorption value, and according to formula：Gastric content residual rate=(absorption values when 20 minutes)/(0 Absorption value during minute), carry out calculating gastric content residual rate.As a result it is as shown in Figure 4.List such as table 4：

4 gastric content residual rate of table determines table

Group	Gastric content residual rate (%)
		Blank control group	57.1±0.2
Positive controls (Pramlintide group)	93.5±1.5
		Test group (Symlin2 groups)	92.4±1.8

From the gastric content residual rate of Fig. 4 and table 4, Pramlintide and Symlin2 groups much larger than sky White matched group, with pole significant difference (P ＜ 0.01), while the gastric content residual rate of Symlin2 groups and Quite, difference is less for Pramlintide group, without significant difference (P ＞ 0.05), shows that Symlin2 suppresses stomach Substantially, i.e. Symlin2 activity in vivo is obvious for the effect of emptying.

Particularly, using this law, the part Pramlintide derivant prepared by the present invention body has been carried out into The detection of interior activity, and result is listed in into table 5：

5 Pramlintide derivant activity in vivo tables of data of table

The test of the blood sugar lowering timeliness of the Pramlintide trim that 15 Pramlintide of embodiment and the present invention are provided

Male and healthy rat (200～250g of body weight) is taken, mice fasting before experiment be can't help water, be randomly divided into 4 groups, 6 per group.The equal insulin injection (0.7U/kg) of every group of rat, therewith by one group of subcutaneous rat therein The isopyknic normal saline of single injection (10ml/kg), its excess-three group difference subcutaneous injection Pramlintide (0.5 Mg/kg), Palmic acid-(γ Glu)-[Lys¹Main chain]-Pramlintide (order is Symlin1,0.6mg/kg) With Palmic acid-(γ Glu)-[Lys¹Side chain]-Pramlintide (order is Symlin2,0.6mg/kg).Injection Tail point blood sampling in 0,0.5,1,2,4,8,12,18,24,36,48 hour afterwards, using Johnson ＆ Johnson of the U.S. The steady person of outstanding talent's type blood glucose meter of company and supporting detection paper blood glucose.Blood glucose value with different time points as vertical coordinate, Time is abscissa, sets up the Time-activity-curve (see Fig. 5) of hypoglycemic activity, calculates each group pharmaceutically-active Biological half-life.As a result shown in table 6：

6 Pramlintide of table, Symlin1 and Symlin2 blood sugar lowering timeliness tables

Sample group	Half-life (h)
		Insulin+normal saline	2.1±0.4
Insulin+Pramlintide	2.5±0.5
		Insulin+Symlin1	13.2±0.4
Insulin+Symlin2	12.9±0.6

As a result show, the drug regimen half-life of insulin+Symlin1 and insulin+Symlin2 compares pancreas Island element+normal saline and insulin+Pramlintide pole dramatically increase (P ＜ 0.01), show modified general Blue woods peptide derivant Symlin1 and Symlin2 blood sugar lowering timeliness is notable compared with Pramlintide.

Particularly, using this law, by the part Pramlintide derivant prepared by the present invention respectively with pancreas Island element combination, has carried out the test of blood sugar lowering timeliness, and the corresponding half-life has been listed in table 7：

7 Pramlintide derivant of table+insulin blood sugar lowering aging effects table

Above-mentioned table data shows that listed compound is that the blood sugar lowering timeliness of modified Pramlintide is extremely notable Better than (P ＜ 0.01) Pramlintide.

The above is only the preferred embodiment of the present invention, it is noted that general for the art For logical technical staff, under the premise without departing from the principles of the invention, some improvement and profit can also be made Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.

Claims

1. a kind of polypeptide, it is characterised in which has the aminoacid sequence shown in (I), (II), (III) In any one：

, and the sequence of (I) or (II) with least 70% homology (III).

2. polypeptide according to claim 1, it is characterised in that the modification include union joint and/ Or fatty acid.

3. polypeptide according to claim 1 and 2, it is characterised in that the union joint selected from γ Glu, Arg、γGlu-Arg、γGlu-His、γGlu-His、Glu-Lys、Glu-Glu、Glu-Arg、γGlu-His-His、 γGlu-Arg-His、γGlu-His-Arg、γGlu-Glu-Arg、Glu-Glu-Arg、Glu-Lys-Arg、 γ Glu-Glu-His-His, γ Glu-Glu-His-Arg, Glu-Glu-Arg-Glu or Glu-Glu-Glu-Glu.

4. the polypeptide according to any one of claims 1 to 3, it is characterised in that (I) has SEQ ID NO:Aminoacid sequence shown in 1；

(I) structure is as shown in formula II：

5. the polypeptide according to any one of Claims 1-4, it is characterised in that described to be substituted by 1,2,3,4 or 5 aminoacid of generation；

The disappearance is 1,2,3,4 or 5 aminoacid of disappearance；

It is described to be added to addition 1,2,3,4,5,6,7,8,9 Or 10 aminoacid.

6. polypeptide according to any one of claim 1 to 5 is preparing treatment diabetes or obesity Application in medicine.

7. a kind of DNA molecular of polypeptide of the coding as described in any one of claim 1 to 5.

8. a kind of recombinant vector, it is characterised in which contains DNA molecular as claimed in claim 7.

9. a kind of host cell, it is characterised in that comprising recombinant vector as claimed in claim 8.

10. a kind of pharmaceutical preparation for treating diabetes and/or obesity, it is characterised in that will by such as right Seek the polypeptide and pharmaceutically acceptable adjuvant composition described in 1 to 5 any one.

A kind of 11. preparation methoies of the polypeptide as described in any one of claim 1 to 5, it is characterised in that Comprise the following steps：

Step 1：Take Tyr and be connected preparation Tyr- resins with resin；

Step 3：By Thr³⁶、Ser³⁴、Thr³⁰、Ser²⁰、Thr⁹Side chain and/or Lys¹Remove-insurance Shield, exposes corresponding functional group's hydroxyl or amino；

Step 4：The union joint is condensed with functional group's hydroxyl or amino, ester bond or amido link is formed Connection；